One of the more unique purines occurring in nature is the base known as Queuine, which is a 7-deaza mammalian cells, Queuine cannot be synthesized by such cells. Instead, Queuine is believed to be obtained from the diet or gastrointestinal microflora as discussed in Nishimura, Proc. Nucleic Acid Res. 28:49-80 (1983). In addition to mammalian systems, Queuine is found widely distributed in nature, including plants, bacteria and other eukaryotic cells such as Tetrahymena and Dictyosterium discoideum as reported in Kasai et al., Nucleic Acids Res. 2:1931-1939 (1975); Farkas, Nucleosides & Nucleotides 2:1-20 (1983); and Katze et al., Science 216:55-56 (1982).
The primary biochemical role of Queuine is reported to be its presence in the wobble position of tRNA that have isoacceptors for tyrosine, histidine, asparagine and aspartic acid. The mechanism for insertion of Queuine at these sites has been well characterized and has been found to occur through the post-transcriptional action of a transglycosylase reaction as described in Okada et al., The Journal of Biological Chemistry 254:3067-3073 (1978); Okada and Nishimura, The Journal of Biological Chemistry 254:3061-3066 (1978).
The role of Queuine in the wobble position of tRNA has not yet been elucidated. However, there is speculation that Queuine may have a role in cell development or as a putative growth factor as discussed in Nishimura, supra: French et al., Proc. Nat'l Acad. Sci U.S.A. 38:370-374 (1991); and Langgut & Kersten, FEBS 265: 33-36 (1990). The role of Queuine may be related to Queuine itself, to its tRNA incorporation or to other Queuine-containing substances. A structural relationship has been suggested with active phorbol ester tumor promoters such as TPA, teleocidin A and phorbol 12,13-didecanoate (PDD). In addition, Queuine has been shown to affect phorbol ester pharmacology. Queuine can also inhibit the activity of 6-thioguanine in repressing cell growth as recently reported in French et al., Proc. Nat'l Acad. Sci. U.S.A. 38:370-374 (1991). Queuine has further been reported to inhibit the differentiation of HL-60 cells by inhibiting the insertion of 6-thioguanine into the wobble position.
Of interest has been the observation that in all animal neoplastic cells examined thus far, including cells transformed in vitro, the tRNAasp, asg, hist, tyr are deficient, or hypomodified, in Queuine. This observation was nicely demonstrated by Randerath et al., Cancer Res. 44: 1167-1171, 1984, who isolated tRNA from normal and from malignant rat liver cells. They completed a full sequence analysis of the tRNAasp and observed that the tRNA from the normal cells had a Queuine molecule in the wobble position, but the wobble position of the tRNA from the malignant cells contained only guanine. These results suggest that Queuine-tRNA (Q-tRNA) deficiency may be tumor specific for a particular tissue. Speculation about the role that Queuine-deficiency might have in mediating the neoplastic state has been made, although a precise function of Q-tRNA has not been identified, and Queuine deficient cells appear to grow normally as reported, for example, in Bienz et al., Nature 294:188-190, 1981).
Although several studies have demonstrated Q-tRNA deficiency in rodent cancers, confirmation that this may occur in undeveloped human tissues has only been suggested in a study by Emmerich et al., Cancer Res. 45:4308-4314, 1985. This study, completed with human lymphomas and chronic stage leukemias, concluded that "decreased Queuine content of tRNA may not be a general feature of neoplasms, but it may be important for disease activity and perhaps also for the state of maturation in human lymphomas and leukemias". The characterization of which types or stages of human cancers are Queuine deficient is of importance and potential therapeutic value.
Another aspect of Queuine biology that has not been characterized is its biodistribution. Mammalian physiological concentrations of Queuine are generally low ranging from 1 to 10 nM with the exception of third trimester bovine amniotic fluid as reported in Katze et al., Supra: Emmerich et al., Cancer Res. 45:4308-4314 (1985). The concentration of Queuine in such late stage bovine amniotic fluid (BAF) is over 100-fold relative to its concentration in fetal bovine serum (FBS) or other body fluids. The high concentration of Queuine in the later stages of BAF additionally suggests that Queuine has a role in embryonal or cell development. However, the mechanism by which the concentration phenomenon is achieved has not been identified.
Studies evaluating the biochemistry of Queuine and Q-tRNA have been limited by the absence of reagents. More particularly, such limitations are related to the relative unavailability of Queuine and the lack of a simple and sensitive detection system for Queuine and Q-tRNA. Queuine must generally be isolated from natural sources that contain low concentrations of Queuine. In addition, the quantitative analysis of Queuine and Q-tRNA has been dependent upon a complicated bioassay described in Katze and Farkas, Proc. Natl. Acad. Sci. USA 76:3271-3275 (1979). Both procedures are limited, difficult and time-consuming. These limitations adversely affect the ability to produce sufficient quantities of Queuine for various purposes and severely limit the scientific evaluation of Queuine and its biological activity.
The availability of a Queuine-specific antibody or binding protein would be of tremendous help in this regard. However, prior to the present invention, antibodies or binding proteins effective for analysis of Queuine have not been produced. As a naturally occurring compound, Queuine does not serve as an effective antigen.
Thus, a need exists for methods of detecting and analyzing Queuine, Queuine-tRNA and other Queuine-containing substances with simple and sensitive assay systems. In addition, methods of obtaining large quantities of Queuine are also needed. The present invention satisfies these needs and provides related advantages as well.